PROJECT SUMMARY Genetic regulation is a fundamental component of all developmental and physiological processes. Conversely, disease states are very often the harmful consequences of disrupted or aberrant gene function. MicroRNAs (miRNA) are ubiquitous small RNAs that constitute a major level of post-transcriptional gene regulation in animals. Indeed, disrupting miRNA genes conserved between humans and mice leads to diverse phenotypes, including defects in the development of the brain, eyes, ears, heart, lungs, muscle, skeleton, teeth, pancreas, intestine, kidneys, liver, breast, testes, and ovaries, accompanied by physiological and behavioral defects and diseases such as epilepsy, deafness, blindness, infertility, autoimmunity, neurodegeneration, diabetes, heart failure, and cancer. On the molecular level, miRNAs function as guides for Argonaute (AGO) proteins, which use the encoded sequence information to identify messenger RNAs (mRNA) targeted for repression. Thus, detailed knowledge of miRNA-target interactions has the potential to illuminate genetic regulatory networks essential to hundreds of developmental, physiological, and disease processes. Despite this extraordinary potential, accurate miRNA-target prediction remains an outstanding challenge, and biochemical methods for capturing miRNA-target interactions are costly and difficult, limiting them to specialized research groups. Thus, despite the enormous opportunity for insight into mammalian biology and discovery of novel drug targets, the vast majority of essential miRNA-target interactions in mammals remain unknown. The objective of this proposal is to create and provide mouse models for robust and reliable miRNA-target discovery that are easy enough to be widely used by diverse researchers. This objective will be achieved by pursuing two specific aims: 1) Identify optimal strategies for tagging AGO2 in mice; and, 2) Develop mouse models for conditional miRNA-target discovery. Under Aim 1 we will adapt recently developed methods, which employ ultra-stable covalent linkages to isolate AGO-RNA adducts from complex molecular mixtures, for the discovery of AGO- RNA interactions mice. We will use our knowledge of AGO structure to devise innovative strategies for encoding tags that allow covalent capture of AGO without compromising AGO function or mouse development. Under Aim 2 we will develop mouse lines that conditionally express tagged forms of AGO that can be used for miRNA-target discovery in specified cell types. This will be a significant advance because it will allow researchers to isolate miRNA-targets involved in discrete physiological processes, and in rare cell types, which is currently not possible. Our rational is that robust, reliable, and accessible tools will allow diverse researchers to discover miRNA-target interactions in processes and diseases relevant to nearly all NIH institutes and centers, as well as greatly advance understanding the biology of these ubiquitous regulatory molecules.